ABSTRACT RA is a chronic inflammatory disorder characterized by progressive joint destruction. Cytokines, especially tumor necrosis factor-? (TNF-?) and interleukin (IL-6), orchestrate the pathogenesis of RA synovitis, and have become important therapeutic targets, but neutralizing antibodies against these cytokines are not always effective. IL-6 is a pleiotropic cytokine that is highly expressed in synovial fluids (SFs) and sera of RA patients. Exogenous soluble IL-6 receptor (IL-6R; CD126) is required by IL-6 to mediate its effects on synovial fibroblasts (FLS), as these cells lack IL-6R. The possible existence of an alternative form of IL-6 that could have powerful actions on FLS has hitherto not been explored. Anti-cyclic-citrullinated peptide antibodies (anti- CCP or ACPA) have become key diagnostic markers for RA, as they are very specific, and are present in approximately 75% of patients with RA. Beyond the role of ACPA in RA, we and others have shown that biological roles of important inflammatory molecules in RA can be altered by citrullination of specific arginine residues. We have now found that IL-6 is citrullinated and, importantly, citrullinated (cit)-IL-6 induces phosphorylation of Erk1/2, Jnk, and Stat3 in RA FLS without requiring the currently known IL-6R. In this novel study, we will investigate the contribution of cit-IL-6 in RA pathogenesis. We will determine the levels of cit-IL-6 and non-cit-IL-6 in RA SFs and serum compared to osteoarthritis (OA) by ELISA. We will examine the role of cit-IL-6 in inducing expression of signaling molecules, cytokines/chemokines, and MMPs in RA FLS and MNs by performing Western blotting, immunofluorescence, ELISAs, and quantitative PCR. We have found that cit- IL-6 induces more MN migration compared to non-cit-IL-6. The role of cit-IL-6 in MN recruitment in RA will be determined by performing MN migration assays with RA SFs depleted of cit-IL-6. We will test the effect of cit- IL-6 in vivo by injecting cit-IL-6 or noncit-IL-6 into mouse knees. We will compare arthritis severity, MN recruitment, and production of pro-inflammatory mediators. To elucidate the mechanism which results in altered functions of cit-IL-6, we will identify the specific arginines converted to citrulline in cit-IL6 by performing liquid chromatography coupled with tandem mass spectrometry. After identifying the specific sites of citrullination, we will generate site directed mutants in which successive arginines will be replaced by another amino acid. IL-6 mutants will be citrullinated and then tested in our panel of signaling and functional assays. We also have data indicating the presence of ACPA against cit-IL-6 in the sera of RA patients. We will further confirm and extend our results using samples from RA and control patients by performing ELISA and dot blot assays using cit-IL-6 as an antigen. This is the first study which demonstrates that cit-IL-6 acts as an inflammatory agonist and autoantigen in RA. These experiments will yield novel insights into the roles of citrullinated IL-6 and autoantibodies to this cytokine in the pathogenesis of RA. Cit-IL-6 may then become a new biomarker and a therapeutic target in RA.